From Homestar Runner Wiki

From time to time, Homestar Runner wears a shirt with a duck on it. In flashback, Strong Badfabricates a history of his first meeting with Homestar Runner (based on the Original Book). In this illustrated story, the duck shirt's exhibition gives rise to several duck-related nicknames for Homestar. In an Easter egg, Homestar dons the shirt from the book to wish Strong Bad a "happy hundredth birthday".

In the DVD commentary for flashback, The Brothers Chaps reveal that the email was originally going to place even more of a focus on Homestar's duckie shirt (which was going to be green, according to Making of Email 100). In the preliminary version, Strong Bad was the original owner of Homestar's star shirt. Marzipan, who did not approve of Homestar's duckie shirt, was to give him the same star shirt as Strong Bad, causing Strong Bad to rip off his shirt in frustration. This was to explain why Strong Bad almost never wears a shirt.

The short, The Homestar Runner Gets Something Stuck In His Craw, reveals what happened to the duckie shirt. Homestar, clad with a duckie shirt, finds something stuck in his craw. It is revealed to be his current shirt, his red shirt with a star on it, which he immediately puts on.

The duckie shirt has made a number of other notable appearances. In Main Page 20 (which is based on flashback), Homestar shows off the duckie shirt's ability to quack. In your funeral, Strong Bad imagines a fat man wearing a duckie shirt who attends his funeral. In Main Page 24, Homestar is again seen wearing the duckie shirt after rolling over "Characters" three times. In the email the paper, Lil' Strong Bad is seen wearing a variation of the Duckie Shirt. It has a red duck with horns and fangs that make it appear to be the devil. In Strong Badia The Free, the flag of Marzistar features the duck from Homestar's duckie shirt on it. For a while, there was even a real-life duckie shirt for sale in the Flash Store.